In 2002, Frommer of Stanford University first developed a FRET biosensor for measuring maltose (Fehr et al., PNAS., 99: 9846-9851, 2002). Since then, other similar types of sensors for measuring ribose (Lager et al., FEBS Lett., 553: 85, 2003), glucose (Fehr et al., J. Biol. Chem., 278: 19127-19133, 2003) or sucrose (Ha et al., Appl. Environ. Microbiol., 73: 7408, 2007) have been continuously developed.
However, the previously developed biosensors show very low detection capabilities, and thus there has been a need to develop highly sensitive sensors which can be used as more accurate measurement means (Fehr et al. Current Opinion in Plant Biology, 7: 345, 2004). In an attempt to satisfy this need, the present inventors previously reported that the detection capability of the FRET biosensor for measuring maltose can be increased by optimizing the linker peptide between the protein domains of the biosensor (Korean Patent Registration No. 10-0739529 (Jul. 29, 2007) and U.S. Pat. No. 7,432,353 (Oct. 7, 2008). Also, Miyawaki (RIKEN Institute) has greatly increased the detection capability of the FRET biosensor “Cameleon” for the measurement of calcium by circular permutation of fluorescent protein (Nagai et al., PNAS., 101: 10554, 2004), and Frommer has increased the detection capability of the FRET biosensor by in-frame fusion of fluorescent protein to QBP (glutamine-binding protein) (Deuschle et al., Protein Sci., 14: 2304, 2006).
However, improving the FRET biosensor by gene manipulation requires many trials and errors, is time-consuming and has reached a technical limit. Thus, a more efficient and easier method for improving the FRET biosensor is required.
Meanwhile, since the three-dimensional structures of proteins were elucidated, studies on the observation of the reversible change in the protein structure and the maintenance of the thermal stability by a change in temperature together with studies on the prediction of the protein folding process have received a great deal of attention from researchers. E. coli periplasmic-binding proteins (PBPs) have become good models for observation of the thermodynamic change of such protein structures. According to a report on the observation of the structural change of arabinose-binding protein (ARBP) depending on a temperature using a differential scanning calorimeter (DSC), reversible unfolding occurred at 53.5° C. in the absence of arabinose, and unfolding occurred at 59° C. in the presence of 1 mM arabinose (Fukuda et al., J. Biol. Chem., 258: 13193. 1983). In addition, it was reported that, for glucose/galactose-binding protein (GGBP), the unfolding temperature increased from 50° C. to 63° C. depending on the presence or absence of glucose (Piszczek et al., Biochem. J., 381: 97, 2004), and for MBP, the unfolding temperature increased by 8-15° C. depending to pH in the presence of maltose (Novokhatny et al., Protein Sci., 6: 141, 1997).
Accordingly, the present inventors have made extensive efforts to the capability of the prior FRET biosensor to measure the concentration of a ligand and to detect a ligand, and as a result, have found that the capability of the biosensor to detect the ligand and measure the ligand concentration is significantly improved when a biosensor consisting of a fusion protein is brought into contact with a ligand at a specific critical temperature at which reversible folding occurs, thereby completing the present invention.